This invention involves the modification of plant developmental responses. Specifically, this invention relates to polynucleotides and polypeptides that affect programmed cell death. These polynucleotides and polypeptides, and genetic constructs comprising such polynucleotides and polypeptides may be used to modulate programmed cell death and thereby alter the developmental cycle of forestry plant cells, hence altering plant development.
Programmed cell death (PCD) refers to an active process, in which gene expression is intimately associated with the events leading to cell death. The plant life cycle contains many instances of such cell death. During plant reproduction and early embryogenesis, events such as organ ablation during unisexual flower development, tapetum degeneration during pollen development and suspensor degeneration during embryo development all involve an active cell death process. During plant morphogenesis and maturation, aleurone cell degradation, the terminal phase of tracheary element differentiation in xylem, leaf blade development in some plants (e.g. genus Monstera), leaf/organ senescence, root cap cell differentiation and the hypersensitive response in plant/pathogen interactions provide further examples of the role of cell death programs in plant developmental cycles.
Most of the scientific investigation relating to programmed cell death to date has involved PCD in mammalian cells. PCD in these cells is evidenced by distinct morphological characteristics, such as cytoplasmic condensation, membrane blebbing, DNA fragmentation, condensation and fragmentation of the nucleus, and finally cell corpse engulfment. In mammalian cells, PCD provides a mechanism for removing unwanted cells, as well as for removing pathogens or pathogen-infected cells. It is also believed that a breakdown in normal PCD mechanisms plays an important role in many disease states, including many malignancies.
The role of PCD in plant systems has not been studied extensively. Preliminary comparisons between plant and mammalian PCD mechanisms suggest some similarities in the mechanisms. The potential similarities include: an oxygen requirement; activation by hydrogen peroxide; a role for calcium in the activation process; a transcription requirement; a dephosphorylation requirement; proteolytic and nucleolytic enzyme involvement and cell condensation and shrinkage. Modulation of the PCD mechanism in any one or more of these areas may affect plant development.
Briefly, the present invention provides isolated polypeptides, and the polynucleotides encoding the isolated polypeptides, having activity in PCD pathways and various developmental pathways in forestry plant species. Genetic constructs comprising such polynucleotides and methods for the use of such genetic constructs to modulate PCD and various developmental pathways in forestry plants are also provided. Transgenic cells and plants incorporating such genetic constructs and exhibiting a modified content of the polynucleotides and/or polypeptides of the present invention compared to a wild-type plant, are also provided. Methods for modulating plant cell death,. as well as for modulating various forestry plant species developmental pathways, using the polynucleotides and/or polypeptides of the present invention, are disclosed.
In mammalian PCD, regulation of cell cycle entry appears to be important, and it has been suggested that cell cycle checkpoint regulators may be involved in the commitment of a cell to death. For example, the known tumor suppressor p45 is capable of mediating cell cycle arrest and can trigger PCD. One of the key genes involved in p45 mediated responses is the retinoblastoma gene (RB). This tumor suppressor can bind and inhibit the transcription factors that initiate entry into the cell cycle. In addition, RB plays a regulatory role in the cell death process, depending on its phosphorylation status. The regulation of RB proteolysis by phosphorylation status, and the consequent RB levels in the cells are important in the determination of cellular fates. Two polynucleotides encoding retinoblastoma-related polypeptides (SEQ ID NOS: 36, 37) have been isolated from forestry species. Retinoblastoma-related polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 80, 81.
Another tumor suppressor gene, prohibitin, can also arrest the cell cycle. In rat B lymphocytes, the association of prohibitin with membrane-bound IgM has been suggested as a mediator of PCD in these cells. Furthermore, in yeast, the deletion of prohibitin homologs resulted in a decreased replicative lifespan, leading to successive decreases in cell cycle time, ageing and cellular senescence. While the above studies have been conducted in non-plant systems, it is likely that similar cell cycle modulators are effective in plant systems. Several polynucleotides encoding prohibitin-related polypeptides (SEQ ID NOS: 22-26) have been isolated from forestry species. Prohibitin-related polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 67-71.
Polynucleotides associated with cellular housekeeping functions are necessary for cell health and survival, and their loss may lead to cell death. One such polynucleotide, initially identified in temperature-sensitive mutant hamster cell lines, was DAD1 (Defender Against Cell Death 1). Cells in temperature-sensitive mutant hamster cell lines undergo PCD at restrictive temperatures, and it has been shown that the Arabidopsis DAD1 can rescue the hamster temperature-sensitive mutant. The presence of DAD1 can also reduce cell death in the developing embryo of the worm Caenorhabditis elegans, which undergoes developmentally-regulated cell death. DAD1 has been shown to be a component of oligosaccharyltransferase, involved in N-linked glycosylation. The induction of cell death by DAD1 inactivation, as well as the ability of DAD1 to reduce PCD during development illustrates the essential role of this housekeeping gene. Several polynucleotides encoding DAD1-related polypeptides (SEQ ID NOS: 6-9) have been isolated from forestry species. DAD1-related polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 51-54.
Another housekeeping polynucleotide which may be used to control cell survival and cell death is the TATA Box Binding Protein (TFIID). TFIID is the most important general factor required for gene transcription by RNA Polymerase II. TFIID binds to the TATA box and participates in the first steps of transcription factor assembly, which is important for the control of gene expression. The ability to developmentally or tissue-specifically knock-out TFIID activity provides a method of specifically inducing cell death. Attempts at TFIID knock-out have not been reported for plants. Polynucleotides encoding TFIID-related transcription initiation factors (SEQ ID NOS: 41, 42) have been isolated from forestry species. TFIID-related transcription initiation factors encoded by the polynucleotides are identified as SEQ ID NOS: 85, 86.
Another transcription factor involved in the control of mammalian cell death is pur-alpha. Pur-alpha is a single-stranded DNA binding protein, which has been shown to play a role in both DNA replication and transcriptional regulation. Pur-alpha is able to suppress PCD of mammalian cells by two mechanisms. The first is the transcriptional repression of Fas (CD-95), a receptor which transduces a cell death signal by interaction with its ligand, and the second is the protection of mammalian cells against cell death mediated by p53. Polynucleotides encoding allelic variants of plant pur-alpha have been isolated (SEQ ID NOS: 90-91) from forestry species. The corresponding amino acid sequences of the pur-alpha polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 141-142.
The actual process of cell death involves the degradation of proteins and nucleic acids, mediated by proteases and nucleases. Experimental work done with mammalian systems suggests that proteases may be an important trigger of cell death. In animals, the caspase family of cysteine proteases are major effectors of this process. Cysteine proteases have been identified in plants which are up-regulated and specifically associated with aleurone and tracheary element cell death. Polynucleotides encoding cysteine proteases in forestry species have been identified as SEQ ID NOS: 92-125. The corresponding amino acid sequences of polypeptides encoded by the polynucleotides are identified by SEQ ID NOS: 143-176. In addition, an aspartic nuclease, nucellin, has been shown to be specifically associated with nucellar cell death. Polynucleotides encoding a nucellin-like aspartic protease (SEQ ID NOS: 15-16) have been isolated from forestry species. The corresponding amino acid sequences of the aspartic nuclease encoded by the polynucleotides are identified in SEQ ID NOS: 60-61.
In addition to actual protease activity, targeting of proteins for proteolytic degradation via the ubiquitin-proteosome pathway is up-regulated during PCD. Human homologs to the Drosophila SINA (Seven In Absentia) gene are activated during PCD. SINA has been shown to target specific proteins for ubiquitination and degradation in both humans and Drosophila. Polynucleotides encoding SINA-related polypeptides (SEQ ID NOS: 38-40, and 200) have been isolated from forestry species. SINA-related polypeptides encoded by the polynucleotides are identified as SEQ ID NOS:. 82-84, and 201.
Nuclear DNA cleavage, nuclear fragmentation and RNA degradation are active processes that occur during PCD in animals and plants. Specific plant DNases and RNases have been identified during PCD in plant aleurone cells, tracheary elements, cells undergoing a hypersensitive response to a pathogen, as well as during salt stress-induced cell death. Polynucleotides encoding a plant DNase (SEQ ID NO: 10) and xylogenic RNase (SEQ ID NO: 45) have been isolated from forestry species. The corresponding amino acid sequences of the DNase and RNase encoded by the polynucleotides are identified in SEQ ID NOS: 55 and 89, respectively.
In mammalian systems, caspase activation can be inhibited by proteins such as Bcl-2, providing protection against cell death. However, other members of the Bcl-2 family, such as Bax, are antagonistic towards the protective effect of Bcl-2 and promote cell death, due to their ability to interact with Bcl-2 and inhibit its protective ability. A recently discovered gene, BI-1 (Bax Inhibitor-1), was found to inhibit Bax-induced cell death. This gene is identical to a previously identified human gene identified as TEGT (Testis Enhanced Gene Transcript). is Polynucleotides encoding TEGT polypeptides isolated from forestry species are identified as SEQ ID NOS: 43-44. The corresponding amino acid sequences of the TEGT polypeptides encoded by the polynucleotides are identified as SEQ ID NOS:87-88.
Another protein involved in inhibition of PCD is BAG-1 (Bcl-2-Associated-athanoGene), a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors in mammalian cells. It is identical to a hormone-receptor binding protein RAP46. BAG-1 binds to and potentiates the effect of the anti-apoptotic protein Bcl-2, to make cells more resistant to apoptosis. Human BAG-1 is overexpressed in human leukemias, colon, cervical, breast, prostate and lung cancer cell lines. A polynucleotide encoding a BAG-1 polypeptide isolated from forestry species is identified as SEQ ID NO: 204. The corresponding amino acid sequence of the BAG-1 polypeptide encoded by the polynucleotide is identified as SEQ ID NO: 205. The isolated polynucleotide sequence encoding BAG-1 contains a PROSITE motif for a ubiquitin-like domain that is also present in the human and mouse BAG-1 proteins.
Numerous studies of mammalian systems have shown that treatments that induce PCD also cause oxidative stress, suggesting a role for oxidative stress in PCD. This has been confirmed by observations that the addition of ROS (Reactive Oxygen Species) or a depletion of cellular antioxidants can cause PCD. PCD can be associated with ROS induction, and PCD can be blocked by the addition of compounds with antioxidant properties. Reactive oxygen species such as superoxide, the hydroxyl radical and hydrogen peroxide can react with and damage cell macromolecules. Additionally, they may set in motion chain reactions in which free radicals are passed from one molecule to another, resulting in extensive cell damage and toxicity.
Plants also exhibit ROS induction during PCD, such as during osmotic stress-mediated death, the hypersensitive response and the terminal stages of tracheary element differentiation. In animal cells, the membrane bound NADPH oxidase complex leads to the generation of superoxide, which is then converted to other ROS. In addition, the small cytosolic protein rac2 is required for activation of the oxidase. When a constitutively active rac2 mutant was inserted into mice, a significant enhancement of PCD occurred compared to wild type mice. Biochemical and immunochemical studies have shown that NADPH oxidase and rac2 are present in plant cells and interact during hypersensitive response PCD. Furthermore, the NADPH oxidase is active during osmotic stress-mediated cell death and during the terminal phase of tracheary element differentiation. The gp 91 NADPH oxidase subunit has been cloned from rice and Arabidopsis. Polynucleotides encoding polypeptides relating to Rac2 (SEQ ID NOS: 28-35) and the gp 91 NADPH oxidase subunit (SEQ ID NO 192) have been isolated from forestry species. The corresponding predicted amino acid sequences for the Rac2-related polypeptides encoded by the polynucleotides given in SEQ ID NOS: 28 and 30-45 are given in SEQ ID NOS: 73 and 74-79. The corresponding predicted amino acid sequence for the gp 91 NADPH oxidase subunit related polypeptide is given in SEQ ID NO: 196.
The role of superoxide compounds in plant cell death was illustrated with the discovery of the lesion simulating cell death (lsd1) mutant in Arabidopsis. In this mutant, superoxide was necessary and sufficient to induce and propagate cell death. Lsd1 in wild type plants is believed to serve as a monitor to a superoxide-dependent signal and to act as a negative regulator of a plant cell death pathway. Polynucleotides encoding lsd1-related polypeptides (SEQ ID NOS: 13 and 14) have been isolated from forestry species. Lsd1-related polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 58 and 59.
ATL2 was identified as an Arabidopsis cDNA which was toxic when overexpressed in yeast. The nucleotide sequences of five ATL2 variants isolated from forestry species are given in SEQ ID NOS: 1-5 and the corresponding predicted amino acid sequences in SEQ ID NOS: 46-50.
Another gene, lls1, identified from a maize mutant, is also required to limit the spread of cell death in a developmental manner in leaves. Polynucleotides encoding lethal leaf spot protein lls1-related polypeptides; (SEQ ID NOS: 11-12) have been isolated from forestry species. Polypeptides encoded by the polynucleotides are identified as SEQ ID NOS: 56-57.
Another plant protein from Arabidopsis (oxy5) has been shown to be a member of the annexin family of proteins and protect bacterial cells from oxidative stress. Oxy5 has also been shown to protect mammalian cells from tumor necrosis factor-induced cell death. The involvement of oxidative stress in the various instances of PCD in plants suggests that oxy5 plays a protective role. The annexin sequences show good homology to oxy5, and hence are expected to provide the same function or similar function. The nucleotide sequences of annexin-like proteins isolated from forestry species are given in SEQ ID NOS: 17-21 and the corresponding predicted amino acid sequences in SEQ ID NOS: 62-66.
The most actively investigated example of PCD in plants concerns the hypersensitive response (HR) to pathogens. The HR is found in most responses mediated by disease resistance (R) genes. The HR is invoked by the association of a pathogen avirulence gene product with a receptor. This sets in motion a cascade of events involving ion fluxes, kinase/phosphatase actions and an oxidative burst leading to localized cell death and the induction of systemic acquired resistance (SAR), in which other parts of the plants develop an acquired resistance to the pathogen. A wide range of plant disease receptors have been identified, including polypeptides that span the cell membrane and contain an extracellular and cytoplasmic domain, as well as polypeptides that are strictly cytoplasmic and do not contain an extracellular domain.
Of the cytoplasmic polypeptide receptors involved in the HR, three families are of primary interest. The first is the RPS2-like polypeptide family, in which the polypeptides include an amino-terminal leucine zipper region, a nucleotide-binding site, an internal hydrophobic domain and a carboxy-terminal leucine-rich repeat. The second is the RPP5-like polypeptide family, in which the polypeptides include an amino-terminal Toll-like domain, a nucleotide-binding site, an internal hydrophobic domain and a carboxy-terminal leucine-rich repeat region. The nucleotide sequences of RPP5-like proteins isolated from forestry species are given in SEQ ID NOS:126-140, and the corresponding predicted amino acid sequences in SEQ ID NOS: 177-191.
The third family of cytoplasmic receptors involved in the HR is the PTO-like family, in which the polypeptides include, a serine-threonine kinase domain. The exact mechanisms by which the HR cell death signals are transduced are not known, although protein-protein interactions and kinase reactions have been shown to be involved in the PTO-like family, with several PTO-interacting protein genes identified.
Downstream of the initial avirulence/receptor interaction, the development of SAR occurs, which involves the NPR1 gene. Mutations in the NPR1 gene increase the susceptibility of plants to pathogen infection and prevent the development of HR PCD and SAR. The expression of R genes in transgenic plants has allowed the development of HR PCD and resistance to specific pathogens. In addition, the expression of PTO-like family members, such as Fen, can lead to PCD in the absence of a pathogen. The nucleotide sequences encoding NPR1-like proteins isolated from forestry species are given in SEQ ID NOS: 193-195 and the corresponding predicted amino acid sequences in SEQ ID NOS: 197-199. The nucleotide sequence encoding a Fen-like protein isolated from forestry species is given in SEQ ID-NO: 27, and the corresponding predicted amino acid sequence in SEQ ID NO: 72. Little is known about the roles of these genes in other cases of plant PCD. An interesting point comes from the realisation that members of the plant R gene families and NPR1 show similarity to several proteins that are involved in animal development and defense. The discovery of a shared pathway linking developmental processes and disease resistance suggests that there may be roles for HR-associated genes in other plant PCD and developmental pathways.
In a first aspect, the present invention provides isolated polynucleotide sequences identified in the attached Sequence Listing as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206; variants of those sequences; extended sequences comprising the sequences set out in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, and their variants; probes and primers corresponding to the sequences set out in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, and their variants; polynucleotides comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206 (x-mers); and extended sequences comprising portions of the sequences set out in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206; all of which are referred to herein, collectively, as xe2x80x9cpolynucleotides of the present invention.xe2x80x9d The present invention also provides isolated polypeptide sequences identified in the attached Sequence Listing as SEQ ID NOS: 46-89, 141-191, 196-199, 201 and 205; polypeptide variants of those sequences; and polypeptides comprising the isolated polypeptide sequences and variants of those sequences.
The polynucleotide sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, were derived from forestry plant sources, namely from Eucalyptus grandis and Pinus radiata. Some of the polynucleotides of the present invention are xe2x80x9cpartialxe2x80x9d sequences, in that they do not represent a full length gene encoding a full length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCR techniques. Partial sequences may be extended until an open reading frame encoding a polypeptide, a full length polynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full length polynucleotides and genes, are described as xe2x80x9ccorresponding toxe2x80x9d a sequence identified as one of the sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, or a variant thereof, or a portion of one of the sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, or a variant thereof, when the extended polynucleotide comprises an identified sequence or its variant, or an identified contiguous portion (x-mer) of one of the sequences of SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, or a variant thereof. Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences, and the like, corresponding to the polynucleotides of the present invention, may be routinely ascertained and obtained using the cDNA sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206.
The polynucleotides identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206, may contain open reading frames (xe2x80x9cORFsxe2x80x9d) or partial open reading frames encoding polypeptides. Additionally, open reading frames encoding polypeptides may be identified in extended or full length sequences corresponding to the sequences set out as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 202 and 206. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, etc. Suitable tools and software for ORF analysis are available for example, on the Internet. Open reading frames and portions of open reading frames may be identified in the polynucleotides of the present invention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, open reading frames encoding polypeptides may be identified using the polynucleotides of the present invention.
Once open reading frames are identified in the polynucleotides of the present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic constructs comprising the open reading frames and suitable promoters, initiators, terminators, etc., which are well known in the art, may then be constructed. Such genetic constructs may be introduced into a host cell to express the polypeptide encoded by the open reading frame. Suitable host cells may include various prokaryotic and eukaryotic cells, including plant cells, mammalian cells, bacterial cells, algae and the like.
Polypeptides encoded by the pplynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively determine levels of interacting proteins or other compounds.
The present invention also contemplates methods for modulating the polynucleotide and/or polypeptide content and composition of a forestry species, such methods involving stably incorporating into the genome of the organism a genetic construct comprising one or more polynucleotides of the present invention. In one embodiment, the target organism is a forestry species, preferably a woody plant, more preferably a woody plant of the Pinus or Eucalyptus species, and most preferably Eucalyptus grandis or Pinus radiata. In a related aspect, a method for producing a forestry plant having an altered genotype or phenotype is provided, the method comprising transforming a plant cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. Forestry plants having an altered genotype or phenotype as a consequence of modulation of the level or content of a polynucleotide or polypeptide of the present invention compared to a wild-type organism, as well as components (seeds, etc.) of such forestry plants, and the progeny of such forestry plants, are contemplated by and encompassed within the present invention.
The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and in positional cloning of genes. Additionally, the polynucleotide sequences identified as SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206, and their variants, may be used to design oligonucleotide probes and primers. Oligonucleotide probes and primers have sequences that are substantially complementary to the polynucleotide of interest over a certain portion of the polynucleotide. Oligonucleotide probes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells using techniques that are well known in the art, such as slot blot DNA hybridization techniques. Oligonucleotide primers designed using the polynucleotides of the present invention may be used for PCR amplifications. Oligonucleotide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the microarray technology used by Synteni (Palo Alto, Calif.).
The polynucleotides of the present invention may also be used to tag or identify an organism or reproductive material therefrom. Such tagging may be accomplished, for example, by stably introducing a non-disruptive non-functional heterologous polynucleotide identifier into an organism, the polynucleotide comprising one of the polynucleotides of the present invention.
The polypeptides of the present invention and the polynucleotides encoding the polypeptides have activity in PCD and various developmental pathways in plants. The polynucleotides were putatively identified by DNA and polypeptide similarity searches. In the attached Sequence Listing, SEQ ID NOS. 1-28, 30-45, 90-140, 192-195, 200 and 204, are polynucleotide sequences that encode the polypeptides listed in SEQ ID NOS. 46-73, 74-89, 141-191, 196-199, 201 and 205, respectively. The polynucleotides and polypeptides of the present invention have demonstrated similarity to the following polypeptides that are known to be involved in PCD and/or plant developmental processes:
In one embodiment, isolated polynucleotides of the present invention comprise a sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206; (b) complements of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206; (c) reverse complements of the sequences recited in SEQ ID NOS: 1-45,;90-140, 192-195, 200, 204 and 206; (d) reverse sequences of the sequences recited in SEQ ID NOS: 1-45, 90-140, 192-195, 200, 204 and 206; and (e) sequences having at least 50%, 75%, 90%, or 98% identity, as defined herein, to a sequence of (a)-(d) or a specified region of a sequence of (a)-(d).
In a further aspect, isolated polypeptides encoded by the polynucleotides of the present invention are provided. In one embodiment, such polypeptides comprise an amino acid sequence recited in SEQ ID NOS: 46-89, 141-191, 196-199, 201 and 205, and variants thereof, as well as polypeptides expressed by polynucleotides of the present invention, including polynucleotides comprising a sequence of SEQ ID NOS: 1-45, 90-140, 192-195, 200 and 204.
In another aspect, the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more additional polynucleotides of the present invention, or in combination with one or more known polynucleotides, together with cells and target organisms comprising such constructs.
In a related aspect, the present invention provides genetic constructs comprising, in the 5xe2x80x2-3xe2x80x2 direction, a gene promoter sequence, an open reading frame coding for at least a functional portion of a polypeptide encoded by a polynucleotide of the present invention, and a gene termination sequence. The open reading frame may be oriented in either a sense or antisense direction. Genetic constructs comprising a gene promoter sequence, a polynucleotide of the present invention, and a gene termination sequence are also contemplated, as are genetic constructs comprising a gene promoter sequence, an untranslated region of a polynucleotide of the present invention, or a nucleotide sequence complementary to an untranslated region, and a gene termination sequence. The genetic construct may further include a marker for the identification of transformed cells.
The gene promoter and termination sequences are preferably functional in a host plant and, most preferably, are those native to the host plant. Promoter and termination sequences that are generally used in the art, such as the Cauliflower Mosaic Virus (CMV) promoter, with or without enhancers such as the Kozak sequence or Omega enhancer, and Agrobacterium tumefaciens nopaline synthase terminator, are useful. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues.
In a further aspect, methods for producing forestry plants having a modified content of a polynucleotide or polypeptide of the present invention compared to a native organism are provided. The methods involve transforming a target forestry plant with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth. Cells comprising the genetic constructs of the present invention are provided, together with tissues and forestry plants comprising such transgenic cells; and fruits, seeds and other products, derivatives, or progeny of such forestry plants.
In yet another aspect of the present invention, methods for modulating PCD, and for modulating various developmental pathways of forestry plants are provided, such methods including stably incorporating into the genome of a forestry plant a genetic construct of the present invention. More specifically, methods for modulating developmental pathways, including wood development, senescence and reproductive development, as well as methods for modulating stress responses in forestry plants, are provided. Preferred forestry plants include woody plants, preferably selected from the group consisting of eucalyptus, pine, acacia, poplar, sweetgum, teak and mahogany species, more preferably from the group consisting of pine and eucalyptus species, and most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. 
The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description.